Icaritin inhibits neuroinflammation in a rat cerebral ischemia model by regulating microglial polarization through the GPER-ERK-NF-κB signaling pathway.

BACKGROUND: Activated microglia play a key role in initiating the inflammatory cascade following ischemic stroke and exert proinflammatory or anti-inflammatory effects, depending on whether they are polarized toward the M1 or M2 phenotype. The present study investigated the regulatory effect of icaritin (ICT) on microglial polarization in rats after cerebral ischemia/reperfusion injury (CI/RI) and explored the possible anti-inflammatory mechanisms of ICT. METHODS: A rat model of transient middle cerebral artery occlusion (tMCAO) was established. Following treatment with ICT, a G protein-coupled estrogen receptor (GPER) inhibitor or an extracellular signal-regulated kinase (ERK) inhibitor, the Garcia scale and rotarod test were used to assess neurological and locomotor function. 2,3,5-Triphenyltetrazolium chloride (TTC) and Fluoro-Jade C (FJC) staining were used to evaluate the infarct volume and neuronal death. The levels of inflammatory factors in the ischemic penumbra were evaluated using enzyme-linked immunosorbent assays (ELISAs). In addition, western blotting, immunofluorescence staining and quantitative PCR (qPCR) were performed to measure the expression levels of markers of different microglial phenotypes and proteins related to the GPER-ERK-nuclear factor kappa B (NF-κB) signaling pathway. RESULTS: ICT treatment significantly decreased the cerebral infarct volume, brain water content and fluorescence intensity of FJC; improved the Garcia score; increased the latency to fall and rotation speed in the rotarod test; decreased the levels of interleukin-1 beta (IL-1ß), tumor necrosis factor-alpha (TNF-α), Iba1, CD40, CD68 and p-P65-NF-κB; and increased the levels of CD206 and p-ERK. U0126 (an inhibitor of ERK) and G15 (a selective antagonist of GPER) antagonized these effects. CONCLUSIONS: These findings indicate that ICT plays roles in inhibiting the inflammatory response and achieving neuroprotection by regulating GPER-ERK-NF-κB signaling and then inhibiting microglial activation and M1 polarization while promoting M2 polarization, which provides a new therapeutic for against cerebral ischemic stroke.

Neuroprotective effects of four different fluids on cerebral ischaemia/reperfusion injury in rats through stabilization of the blood-brain barrier.

Protecting the blood-brain barrier (BBB) is a potential strategy to treat cerebral ischaemic injury. We previously reported that hypertonic sodium chloride hydroxyethyl starch 40 (HSH) treatment alleviates brain injury induced by transient middle cerebral artery occlusion (tMCAO). However, other fluids, including 20% mannitol (MN), 3% hypertonic sodium chloride (HTS) and hydroxyethyl starch 130/0.4 solution (HES), have the same effect as HSH in cerebral ischaemia/reperfusion injury (CI/RI) remains unclear. The present study evaluated the protective effects of these four fluids on the BBB in tMCAO rats. Sprague-Dawley (SD) rats were randomly assigned to six groups. A CI/RI rat model was established by tMCAO for 120 min followed by 24 h of reperfusion. The sham and tMCAO groups were treated with normal saline (NS), whereas the other four groups were treated with the four fluids. After 24 h of reperfusion, neurological function, brain oedema, brain infarction volume, permeability of the BBB, cortical neuron loss and protein and mRNA expression were assessed. The four fluids (especially HSH) alleviated neurological deficits and decreased the infarction volume, brain oedema, BBB permeability and cortical neuron loss induced by tMCAO. The expression levels of GFAP, IL-1ß, TNF-α, MMP-9, MMP-3, AQP4, MMP-9, PDGFR-ß and RGS5 were decreased, whereas the expression levels of laminin and claudin-5 were increased. These data suggested that small-volume reperfusion using HSH, HES, MN and HTS ameliorated CI/RI, probably by attenuating BBB disruption and postischaemic inflammation, with HSH exerting the strongest neuroprotective effect.

Icaritin alleviates cerebral ischemia‒reperfusion injury by regulating NMDA receptors through ERK signaling.

N-methyl-D-aspartate (NMDA) receptors are key signaling molecules that mediate excitotoxicity during cerebral ischemia. GluN2A-containing NMDA receptors, which are mostly located in the intrasynaptic region, mediate normal physiological processes and promote neuronal survival. GluN2B-containing NMDA receptors, which are mostly located in the extrasynaptic region, mediate excitotoxicity injury and promote neuronal death during ischemia. This study investigated the ability of icaritin (ICT) to protect against cerebral ischemia‒reperfusion injury (CI/RI) by regulating GluN2B-containing NMDA receptors through extracellular signaling regulatory kinases/death associated protein kinase 1 (ERK/DAPK1) signaling. A rat CI/RI model was established by transient middle cerebral artery occlusion (tMCAO). Following treatment with ICT and the ERK-specific inhibitor U0126, cerebral infarction, neurological function, and excitotoxicity-related molecule expression were assessed 24 h after reperfusion. ICT treatment significantly decreased cerebral infarct volume, improved neurological function, and regulated NMDA receptor subtype expression and ERK/DAPK1 signaling activation. The ability of ICT to increase GluN2A and postsynaptic density protein 95 (PSD95) mRNA and protein expression, inhibit GluN2B expression, and regulate DAPK1 activation was reversed after administration of the ERK-specific inhibitor U0126. These data indicated that ICT inhibited excitotoxicity injury and exerted a protective effect against CI/RI that was likely mediated by increased ERK signaling pathway activation and regulation of extrasynaptic and intrasynaptic NMDA receptor function, providing a new therapeutic target for ischemic encephalopathy.